Interferon (IFN)-gamma is a prominent activator of pathways that effect host defense against intracellular pathogens, including many category A, B, and C biodefense priority pathogens. The Immunity-related GTPases (IRG; also known as p47 GTPases) are a family of IFN-gamma-induced proteins that play critical roles in resistance to intracellular bacteria and protozoa, including Salmonella typhimurium and Toxoplasma gondii. Mice lacking certain IRG proteins have crippled host defense against these pathogens; further, macrophages isolated from the IRG-deficient mice display defects in phagosome processing and intracellular killing, as well as in cellular motility. However, the underlying mechanisms are unknown. Based on our published and preliminary data, we hypothesize the following: Hypothesis 1. IRG proteins form complexes among themselves - both homotypic and heterotypic - that regulate phagosome processing and macrophage motility. Hypothesis 2. IRG proteins in the GMS subfamily play dominant roles as regulators of the IRG proteins in the GKS subfamily. GMS proteins complex with specific GKS proteins to prevent their degradation, enabling subsequent GKS protein assembly in homotypic complexes at sites of contact between lipid membranes and the actin cytoskeleton, impacting phagosome processing and cell motility. We will address these hypotheses with the following: Aim 1. We will define homotypic and heterotypic IRG protein complexes that exist among specific GMS and GKS proteins in activated macrophages. We will determine the localization of the complexes, and the time frames in which they appear as related to cell motility and phagosome processing. We will determine the dependence on biochemical activity and protein domains on formation of the complexes. Aim 2. We will define the role of IRG GMS proteins in regulating IRG GKS proteins, and in coordinating IRG protein-mediated host defense functions. We will determine the effect of GMS protein deficiency on regulating GKS protein levels. Where levels are altered, we will determine whether GMS proteins impact GKS expression at the level of transcription, translation, and/or protein stability. We will determine the effect of IRG GMS protein-deficiency on the creation of homotypic IRG GKS protein complexes on the phagosome, and on the plasma membrane at the leading edge of the migrating macrophages. PUBLIC HEALTH RELEVANCE: This research will elucidate a fundamental mechanism that governs resistance to intracellular bacteria and protozoa. Our ultimate goals are the generation of broadly applicable therapeutic agents for human infectious disease and biodefense.